Giuseppina Mignogna

Antimicrobial peptides from amphibian skin: What do they tell us?

Amphibian skin secretions contain many biologically active compounds, such as biogenic amines, complex alkaloids, or peptides. Within the latter class of molecules, a large number of peptide antibiotics has been isolated and characterized from different amphibian species. Antimicrobial peptides are considered the effector molecules of innate immunity, acting as a first line of defense against bacterial infections, by perturbing the phospholipid bilayer of the target cell membrane. These gene-encoded molecules are synthesized as inactive precursors and in several cases their proparts were shown to have highly conserved structures. It has also been demonstrated that the promoter regions of inducible peptide antibiotics are often regulated by the transcriptional control machinery NF-kappa B/I kappa B alpha. In amphibia of Rana and Bombina genera, inhibition of transcription of the genes encoding antimicrobial peptides has been obtained by glucocorticoid treatment, which causes an increase of I kappa B alpha synthesis. Moreover, determination of the structure of a number of genes coding for antimicrobial peptides in amphibia has actually shown that their promoter regions contain recognition sites for nuclear factors.

A Novel Non-heme Iron-binding Ferritin Related to the DNA- binding Proteins of the Dps Family in Listeria innocua*

A multimeric protein that behaves functionally as an authentic ferritin has been isolated from the Gram-positive bacterium Listeria innocua The purified protein has a molecular mass of about 240,000 Da and is composed of a single type of subunit (18,000 Da). L. innocua ferritin is able to oxidize and sequester about 500 iron atoms inside the protein cage. The primary structure reveals a high similarity to the DNA-binding proteins designated Dps. Among the proven ferritins, the most similar sequences are those of mammalian L chains that appear to share with L. innocua ferritin the negatively charged amino acids corresponding to the iron nucleation site. In L. innocua ferritin, an additional aspartyl residue may provide a strong complexing capacity that renders the iron oxidation and incorporation processes extremely efficient. This study provides the first experimental evidence for the existence of a non-heme bacterial ferritin that is related to Dps proteins, a finding that lends support to the recent suggestion of a common evolutionary origin of these two protein families.

Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model. Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells. Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Novel antimicrobial peptides from skin secretion of the European frog Rana esculenta

Three antimicrobial peptides were isolated from skin secretion of the European frog, Rana esculenta. Two of them show similarity to brevinin‐1 and brevinin‐2, respectively, two antimicrobial peptides recently isolated from a Japanese frog [Morikawa, N., Hagiwara, K. and Nakajima, T. (1992) Biochem. Biophys. Res. Commun. 189, 184‐190]. The third one, named esculentin, is 46 residues long and represents a different type of peptide. All these peptides have as a common motif an intramolecular disulfide bridge located at the COOH‐terminal end. The peptides from R. esculenta show distinctive antibacterial activity against representative Gram‐negative and Gram‐positive bacterial species. In particular, esculentin is the most active against Staphylococcus aureus, and has a much lower hemolytic activity.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography–tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 μg/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue β2-AR (β2 adrenergic receptor) and white adipose tissue (WAT) PPAR-δ (peroxisome proliferator-activated receptor δ), β3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Effects of Mentha suaveolens Essential Oil Alone or in Combination with Other Drugs in Candida albicans

Candidosis is the most important cause of fungal infections in humans. The yeast Candida albicans can form biofilms, and it is known that microbial biofilms play an important role in human diseases and are very difficult to treat. The prolonged treatment with drugs has often resulted in failure and resistance. Due to the emergence of multidrug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to analyse the effects induced by essential oil of Mentha suaveolens Ehrh (EOMS) on Candida albicans and its potential synergism when used in combination with conventional drugs. Morphological differences between control and EOMS treated yeast cells or biofilms were observed by scanning electron microscopy and transmission electron microscopy (SEM and TEM resp.,). In order to reveal the presence of cell cycle alterations, flow cytometry analysis was carried out as well. The synergic action of EOMS was studied with the checkerboard method, and the cellular damage induced by different treatments was analysed by TEM. The results obtained have demonstrated both the effects of EOMS on C. albicans yeast cells and biofilms and the synergism of EOMS when used in combination with conventional antifungal drugs as fluconazole (FLC) and micafungin (MCFG), and therefore we can hypothesize on its potential use in therapy. Further studies are necessary to know its mechanism of action.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin

Gene‐encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF‐κB is an important transcription factor for many immunity‐related mammalian proteins and also for insect immune genes. The activity of NF‐κB is regulated by the interaction with an inhibitor, IκB. It was recently demonstrated that glucocorticoids induce the synthesis of IκB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF‐κB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of IκBα. These results suggest that also in vertebrates peptide‐mediated innate immunity is controlled by NF‐κB‐regulated transcription.

An amphibian antimicrobial peptide variant expressed in Nicotiana tabacum confers resistance to phytopathogens.

Esculentin-1 is a 46-residue antimicrobial peptide present in skin secretions of Rana esculenta. It is effective against a wide variety of micro-organisms, including plant pathogens with negligible effects on eukaryotic cells. As a possible approach to enhance plant resistance, a DNA coding for esculentin-1, with the substitution Met-28Leu, was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein, under the control of the cauliflower mosaic virus 35S promoter region, and introduced into Nicotiana tabacum. The antimicrobial peptide was isolated from the intercellular fluids of healthy leaves of transgenic plants, suggesting that it was properly processed, secreted outside cells and accumulated in the intercellular spaces. The morphology of transgenic plants was unaffected. Challenging these plants with bacterial or fungal phytopathogens demonstrated enhanced resistance up to the second generation. Moreover, transgenic plants displayed insecticidal properties.

Structure-function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions☆

Skin secretions of amphibia of the Bombina genus contain two families of antimicrobial peptides, the bombinins (bombinin-like peptides) and the bombinins H (H for hydrophobic and hemolytic). The latter family includes a number of peptides containing a D-amino acid in the second position, in addition to their corresponding all L-isomers. The antimicrobial activity of three pairs of bombinin H isomers, H2/H4, H6/H7 and GH-1D/GH-1L, has been investigated. The first two pairs of peptides were actually isolated from the secretion, whereas the third was synthesized according to the sequence deduced from a gene coding for a bombinin-like peptide in Bombina orientalis.